Periodic Report- Pathfinder
Nitsara Karoonuthaisiri
1. Final Publishable Summary Report (2 pages)
Background
Access to sufficient, safe & wholesome food has been man's main endeavor from the
earliest days of human existence and is one of the basic human rights. One of the main
challenges in delivering a safe food supply is the early detection of pathogens to prevent
illness due to the consumption of contaminated foods. Pathfinder is an important research
project focused on development of innovative means of detecting important pathogenic
organisms associated with food poisoning. Molecular binders such as monoclonal antibodies
and phage-derived binders were developed and screened for specificity to important
foodborne pathogens such as Salmonella and Listeria monocytogenes. The technology
platforms were evaluated for their capability of detecting many different pathogens
simultaneously by using Salmonella spp., Listeria monocytogenes, and Campylobacter as a
model. The project was proposed into two phases: Incoming phase and Returning phase as
depicted in Fig. 1. This report focuses only on Work Package 4, which was conducted during
the Returning phase.
Work Package 1
Antibody Development
Work Package 2
Multiplex Selection
Work Package 4
Comparison of multiplex performance
Incoming Phase
Fig. 1. Overview of resarch program for Pathfinder
Overview of Results
Previously, during the incoming phase, a catalog of highly specific monoclonal
antibodies to Listeria monocytogenes has been produced and screened by high-throughput
screening methods based on microarray and Surface Plasmon Resonance (SPR) [1]. In
addition to the proposed production of antibodies, two libraries of bacteriophages were
screened for an alternative binder for Salmonella and Listeria monocytogenes. Several phage
peptides demonstrated highly specific binding characteristics to the targets and have been
applied to magnetic separation as a detection method [2, 3]. These specific binders and other
available binders were employed to evaluate different biosensor platforms such as SPR [4, 5],
microcantilever [6], and a bead-array technology (MAGPIX) [7]. After such evaluation, a
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bead-array technology was the most appropriate method due to its multiplex capacity and
sensitivity which were better than the standard ELISA method.
During the returning phase, the bead-array technology was used to perform multiplex
detection of Salmonella spp., Listeria monocytogenes, and Campylobacter spp. in actual food
samples (roasted chicken barbecue with honey, grilled black pepper chicken, and raw chicken
marinated in sesame sauce) [8].
To simulate foodborne pathogens contamination, each pathogen was inoculated into
the food samples at 0, 1, and 10 colony forming unit (CFU). The samples were subsequently
cultured according to the international organization for standardization (ISO 10272, 11290,
and 6579 for Campylobacter spp., Listeria monocytogenes, and Salmonella spp.,
respectively). Samples from each culture protocol were mixed and tested at 24, 48 and 72
hours by the bead-array method and a conventional plate-count protocol to enumerate the
bacteria number. The results showed that the bead-array method was able to detect even at 1
CFU after being cultured for 24 hours in case of Salmonella spp. and 48 hours in case of
Listeria monocytogenes and Campylobacter spp. From the plate-count method of the 24-hr
culture of Salmonella spp and the 48-hr cultures of Listeria monocytogenes and
Campylobacter spp., 6.0- 40.0 x 107 CFU/mL, 5.0-10.0 x 108 CFU/mL and 2.0-6.0 x 108
CFU/mL were found, respectively.
Conclusion
Pathfinder project has considerably made advancement for the food safety measure
by constructing a novel multiplex detection method for foodborne pathogens. Through the
process of the development, this project generated several important research milestones.
First, different types of binders, monoclonal antibodies and phage-derived peptides, were
produced, screened and exploited for their molecular binding capacity. These developed
bioreceptors from this project can be further applied to other types of biosensor platforms or
other applications beyond imagination. Second, several technology platforms were evaluated.
Through these evaluations, multiplex detections for both food and plant pathogens were
achieved. The optimization and signal enhancement strategies can also serve as a guideline
for similar assay development for these types of biosensors. In addition, the developed bead
array method has been demonstrated to be able to detect Campylobacter spp., Listeria
monocytogenes, and Salmonella spp. contaminated in various actual food samples using the
conventional enrichement methods according to the international organization for
standardization.
Socio-economic Impacts
The research outputs are of direct significance to food producers/processors, food inspectors
and regulatory and testing laboratories. A successful validation of the developed multiplex
detection based on bead-array technology is to be transferred to food producers. This will
provide a rapid, accurate, economical testing method for the food industry to reduce the
testing cost while increase the credibility for food safety. Moreover, the ability to prevent
bacterial contamination in food chain means betterment for public health.
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